The long-term goals of this proposed research are to understand the mechanisms governing degenerative processes in aging, Alzheimer's disease, and other age-related pathologies. Specifically, this project focuses on an enzyme (calpain) whose substrates include brain spectrin and the neurofilament and microtubule-associated proteins. Calpain is a calcium-activated neutral protease that has been implicated in both experimentally- induced and disease-related degeneration. It is present in neurons that are particularly susceptible to age-related pathology, and its activity in brain is linearly and negatively correlated with the maximal life span of eight orders of mammals. The specific aims of this proposed research are (1) to compare the cellular and subcellular distribution of calpain and three of its substrates, brain spectrin, the 200 kD neurofilament protein, and microtubule-associated protein 2 in young adult, mid-life, and elderly rats, (2) to localize calpain and the abovementioned substrates in human autopsy material from patients with Alzheimer's disease, amyotrophic lateral sclerosis, multiple infraction dementia and normal aging at the light and electron microscopic levels, (3) to determine whether calpain is located in or near neurofibrillary tangles or neuritic plaques, (4) to evaluate whether there is a selective loss of calpain containing cells in Alzheimer's disease, and (5) to test whether calpain's activity, or the concentration of the enzyme or its substrates, is altered in age or age-related disease. Thus, this proposed research seeks to determine the role of calpain in the cellular degeneration that accompanies age and pathology. To accomplish this end, light and electron microscopic examination of immunostained tissue, biochemical assay of enzyme activity, and immunoblots will be used. These studies should significantly add to our understanding of the mechanisms involved in the cytoskeletal disarray seen with age and age-related pathologies, as well as to delineate differences and similarities between these different pathologies.